The insulin receptor (IR) is a heterotetrameric glycoprotein composed of two alpha and two beta subunits linked by disulfide bonds. The alpha subunit is extracellular and binds insulin, while the beta subunit is transmembranal and functions as a cytosolic tyrosine protein kinase. Both alpha and beta subunits are coded for by a single mRNA whose gene is located on Chromosome 19p. The overall objective of this proposal is to study how inherited variations in the alpha subunit of the human IR gene alter cell physiology. To accomplish this goal we have identified families whose probands express monogenic, severe, insulin resistance, hypoglycemia, and leprechaunism. When insulin binding to their cultured skin fibroblasts is used as a genetic discriminant, an autosomal recessive pattern of inheritance emerges in which probands' cells have specific absence of high-affinity, insulin binding and parents' cells have partial impairment. These observations imply mutations in the IR alpha subunits. Glucose transport by cells from all probands is insulin-insensitive but one patient's cells have markedly increased, basal glucose transport. We will test the hypothesis that different mutations in the IR alpha subunit are the cause for those phenotypes of insulin resistance and loss of glucose homeostasis. To this end we will evaluate the structure of normal and mutant IR using affinity crosslinking of 125I-insulin to alpha subunits, 32P-ATP incorporation into beta subunits, and immunoblotting of the intact and disassociated IR. We will compare cellular signaling using insulin-enhanced, IR autophosphorylation, exogenous protein phosphorylation, and regulation of membrane transport. To clarify the relationship between increased, insulin-insensitive glucose uptake and mutations in the IR alpha subunit, we will transfect this unique proband's fibroblasts with normal IR cDNA. When insulin binding is normalized, we will evaluate glucose transporter function synthesis, turnover and gene expression. Transmission of the IR gene in these families will be explored by RFLP analysis. Preliminary RFLP analyses identified rare variations in the IR alpha subunit DNA which were transmitted with Mendelian patterns but revealed no gross deletions or insertions. Similarly normal and mutant IR mRNAs had the same size and quantity. We will procede to identify presumptive base pair substitutions in the alpha gene using ribonuclease cleavage at RNA:DNA mismatches. The area of interest of the mutant cDNA will be cloned and sequenced. In this way we will develop molecular methods of predicting inherited insulin resistance and clarify a novel signal by which the IR alpha subunit may regulate glucose transporter activity.